New Insights into Genetic Diversity and Differentiation of 11 Buffalo Populations Using Validated SNPs for Dairy Improvement

<b>Background/Objectives:</b> Buffalo populations exhibit distinct genetic variations influenced by domestication history, geographic distribution, and selection pressures. This study investigates the genetic structure and differentiation of 11 buffalo populations, focusing on five loci related to milk protein (<i>CSN1S1</i> and <i>CSN3</i>) and fat metabolism (<i>LPL, DGAT1</i> and <i>SCD</i>). The aim is to assess genetic variation between river, swamp, and wild-type buffaloes and identify key loci contributing to population differentiation. <b>Methods:</b> Genetic diversity was analyzed through allele frequency distribution, the Hardy-Weinberg equilibrium testing, and observed (Ho) and expected heterozygosity (He) calculations. Population structure was assessed using principal component analysis (PCA), F_ST statistics, and phylogenetic clustering (k-means and UPGMA tree). The silhouette score (SS) and the Davies-Bouldin index (DBI) were applied to determine optimal population clustering. <b>Results:</b> Significant genetic differentiation was observed between river and swamp buffaloes (<i>p</i> < 0.001). <i>DGAT1</i> and <i>CSN3</i> emerged as key markers distinguishing buffalo types. The Italian Mediterranean buffalo exhibited the highest genetic diversity (Ho = 0.464; He = 0.454), while the Indonesian, Chinese, and Vietnamese populations showed low heterozygosity, likely due to selection pressures and geographic isolation. The global F_ST (0.2143; <i>p</i> = 0.001) confirmed moderate differentiation, with closely related populations (e.g., Nepal and Pakistan) exhibiting minimal genetic divergence, while distant populations (e.g., Egypt and Indonesia) showed marked differences, and the Romanian population showed a unique genetic position. <b>Conclusions:</b> These findings contribute to a deeper understanding of buffalo genetic diversity and provide a valuable basis for exploiting the potential of this species in the light of future breeding and conservation strategies specific for each buffalo type.